Elucidating Extracellular Matrix and Stiffness Control of Primary Human Hepatocyte Phenotype via Cell Microarrays

Abstract

the protein composition and stiffness of the ECM regulate the functions of human liver cells in physiology and disease has not been fully elucidated, and doing so in vivo in rodents is challenging due to the presence of several confounding variables (e.g., fluid flow, nonparenchymal interactions, and soluble factors) and speciesspecific differences in liver functions. [5] Therefore, to mitigate the limitations with animal studies, cultures of primary human hepatocytes (PHHs) are used routinely for drug screening, disease modeling, and mechanistic inquiries. [6] However, PHHs display a precipitous decline in phenotypic functions when cultured on collagen-I adsorbed onto tissue culture polystyrene (TCPS) and glass. [7] Sandwiching hepatocytes within two layers of gelled collagen-I can induce the reformation of bile canaliculi but other functions still show rapid decline, [8] potentially due to an excessive amount of collagen-I that is more akin to liver fibrosis. Similarly, culturing hepatocytes with tumor-derived murine Matrigel can induce some functions for ≈1 week, [7] though comparisons to human liver ECM are challenging. In contrast, culturing PHHs on decellularized human liver ECM can also transiently improve phenotypic functions; [9] however, such ECM is typically variable in quality due to the unpredictable conditions of the transplant-rejected human livers and the harsh cellular dissociation methods. [10] In contrast to complex ECM gels such as those discussed above, recombinant and purified ECM proteins can be useful to elucidate how such proteins affect the hepatic phenotype individually and in specific combinations, often in unexpected ways. Previously, rat hepatocytes cultured on different combinations of recombinant ECM proteins for 7 d displayed varying albumin staining on ECM combinations, and such differences were often due to the unexpected interactions between different ECM proteins. [11] In addition to the protein composition of the ECM, the hepatocyte phenotype in vitro is highly sensitive to the underlying substrate's stiffness. Specifically, primary mouse hepatocytes, [3] primary rat hepatocytes, [12,13] mouse embryonic stem cell (ESC)-derived-hepatocytes, [14] PHHs, [15] and human ESC-derived hepatocytes [16] all displayed higher levels of phenotypic functions when cultured on softer ECM gels versus stiffer substrates. However, how the protein composition and stiffness How the liver's extracellular matrix (ECM) protein composition and stiffness cooperatively regulate primary human hepatocyte (PHH) phenotype is unelucidated. Here, protein microarrays and high-content imaging with single-cell resolution are utilized to assess PHH attachment/functions on 10 major liver ECM proteins in single-and two-way combinations robotically spotted onto polyacrylamide gels of 1 or 25 kPa stiffness. Albumin, cytochrome-P450 3A4 (CYP3A4), and hepatocyte nuclear factor alpha (HNF4α) positively correlate with each other and cell density on both stiffnesses. The 25 kPa stiffness supports higher average albumin a...

An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

“…The liver is a highly vascularized soft organ and performs various vital functions in the human body such as homeostasis, bile acid production, detoxification, and drug metabolism [ 1 ]. In the process of drug metabolism, liver is often exposed to the adverse effects of many drugs.…”

Decellularized Liver Nanofibers Enhance and Stabilize the Long‐Term Functions of Primary Human Hepatocytes In Vitro

“…In contrast to albumin secretion, PHH urea synthesis (a widely utilized marker of liver ammonia detoxification) rates displayed a continuous decline over time in all cultures (nanofibers and adsorbed ECM control substrates) and after 23 days had decreased by 90–98% of day 1 levels; however, urea synthesis rates were still 2.5‐fold higher on the collagen nanofibers, 1.03‐fold higher on the collagen/chitosan nanofibers, 1.8‐fold higher on the collagen/PLECM nanofibers, 5.3‐fold higher on the PLECM nanofibers, and 1.7‐fold higher on the PLECM/chitosan nanofibers as compared to the adsorbed ECM controls after 23 days of culture (Figure 5B). Urea synthesis involves the coordinated action of several enzymes of the urea cycle and is typically difficult to maintain at steady levels in PHH monocultures even with various ECM combinations/manipulations; [ 10 ] therefore, it was not surprising here that nanofibers upregulated but did not stabilize urea synthesis in PHH monocultures as compared to adsorbed ECM substrates.…”

“…The results above show that while nanofibers can upregulate the activities of several different CYP isoforms as compared to adsorbed ECM substrates, they are unable to stabilize CYP levels for several weeks, which suggests that ECM manipulations alone are necessary but not sufficient to maintain CYP activities in PHHs over prolonged culture; we and others have previously seen something similar with other substrates in which ECM was the only microenvironmental manipulation for PHH monocultures in vitro. [3,7,10]…”

Engineered matrix microenvironments reveal the heterogeneity of liver sinusoidal endothelial cell phenotypic responses

“…Given the considerable evidence that LSEC behavior is strongly influenced by both ECM composition and stiffness independently, 23,27 we sought to better understand how these two microenvironmental parameters, in various, physiologically relevant combinations, could further affect LSEC phenotype. Using a cellular microarray platform, we selected 28 ECM combinations previously implicated in influencing hepatic cell phenotype from literature, 21,23,35 and three different hydrogel stiffnesses that mimic the mechanical properties of different stages of liver fibrosis: 1 kPa for healthy tissue, 6 kPa for early-stage fibrosis, and 25 kPa for late-stage fibrosis. 41,42 In total, we tested 84 unique combinatorial microenvironments for their impact on LSEC phenotype.…”